Limits...
RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition.

Newhart A, Powers SL, Shastrula PK, Sierra I, Joo LM, Hayden JE, Cohen AR, Janicki SM - Mol. Biol. Cell (2016)

Bottom Line: Rpp29 is a protein subunit of RNase P.Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly.Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Wistar Institute, Philadelphia, PA 19104.

No MeSH data available.


Related in: MedlinePlus

Rpp29 represses histone H3.3 chromatin assembly. (A) Western blot shows levels of endogenous H3.3 and H3.3-YFP in the HeLa HI 1-1/H3.3-YFP (lane 1) and HeLa HI 1-1 (lane 2) cell lines. (B) Diagram of the steps undertaken in the high-salt extraction and chromatin immunoprecipitation protocols. (C) qRT-PCR and Western blot analysis of Rpp29 mRNA and ATRX protein levels, respectively, in HeLa HI 1-1 cells after shRNA knockdown. For qRT-PCR, results are the average of three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (D) The transgene diagram shows the location of the primer pairs used for qPCR analysis of the ChIP results in HeLa HI 1-1/H3.3-YFP cells. (E) ChIP analysis of H3.3-YFP incorporation into the transgene array after knockdowns in HeLa HI 1-1/H3.3-YFP cells. Results are the average of at least three independent experiments, and p values were calculated by comparing pLKO.1 (blue bar) to the shRNA data sets using unpaired t test: *p ≤ 0.05 and **p ≤ 0.01. (F) Representative Western blot of high-salt extraction assay showing H3.3-YFP and H3.3 levels detected using anti-GFP and anti-H3.3 antibodies. Graphs show measurements of (I) H3.3-YFP with anti-GFP antibody, (II) H3.3-YFP with anti-H3.3 antibody, and (III) H3.3 with anti-H3.3 antibody. Results are the average of at least three independent experiments, and p values were calculated using unpaired t test: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (G) ChIP analysis of H3.3-YFP incorporation into the transgene array in HeLa HI 1-1/H3.3-YFP cells after first detergent extracting and vortexing isolated nuclei.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4814222&req=5

Figure 8: Rpp29 represses histone H3.3 chromatin assembly. (A) Western blot shows levels of endogenous H3.3 and H3.3-YFP in the HeLa HI 1-1/H3.3-YFP (lane 1) and HeLa HI 1-1 (lane 2) cell lines. (B) Diagram of the steps undertaken in the high-salt extraction and chromatin immunoprecipitation protocols. (C) qRT-PCR and Western blot analysis of Rpp29 mRNA and ATRX protein levels, respectively, in HeLa HI 1-1 cells after shRNA knockdown. For qRT-PCR, results are the average of three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (D) The transgene diagram shows the location of the primer pairs used for qPCR analysis of the ChIP results in HeLa HI 1-1/H3.3-YFP cells. (E) ChIP analysis of H3.3-YFP incorporation into the transgene array after knockdowns in HeLa HI 1-1/H3.3-YFP cells. Results are the average of at least three independent experiments, and p values were calculated by comparing pLKO.1 (blue bar) to the shRNA data sets using unpaired t test: *p ≤ 0.05 and **p ≤ 0.01. (F) Representative Western blot of high-salt extraction assay showing H3.3-YFP and H3.3 levels detected using anti-GFP and anti-H3.3 antibodies. Graphs show measurements of (I) H3.3-YFP with anti-GFP antibody, (II) H3.3-YFP with anti-H3.3 antibody, and (III) H3.3 with anti-H3.3 antibody. Results are the average of at least three independent experiments, and p values were calculated using unpaired t test: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (G) ChIP analysis of H3.3-YFP incorporation into the transgene array in HeLa HI 1-1/H3.3-YFP cells after first detergent extracting and vortexing isolated nuclei.

Mentions: To determine whether Rpp29 regulates H3.3 nucleosome deposition, we knocked Rpp29 down in the HeLa cell line stably expressing H3.3-YFP (Figure 8A) and evaluated H3.3 incorporation into the inactive transgene array using chromatin immunoprecipitation (ChIP; Figure 8B is a diagram of the procedure). We previously reported that, in the HeLa cell line, H3.3 is significantly enriched in the inactive transgene array (Figure 8D is a diagram of the primer pairs) compared with the RC variant, H3.2 (Newhart et al., 2012). Here we show that Rpp29 knockdown significantly increases H3.3 incorporation into the array (Figure 8E, brown bars). Taken together, these results suggest that Rpp29 represses H3.3 chromatin assembly by down-regulating a transcriptional recruitment signal.


RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition.

Newhart A, Powers SL, Shastrula PK, Sierra I, Joo LM, Hayden JE, Cohen AR, Janicki SM - Mol. Biol. Cell (2016)

Rpp29 represses histone H3.3 chromatin assembly. (A) Western blot shows levels of endogenous H3.3 and H3.3-YFP in the HeLa HI 1-1/H3.3-YFP (lane 1) and HeLa HI 1-1 (lane 2) cell lines. (B) Diagram of the steps undertaken in the high-salt extraction and chromatin immunoprecipitation protocols. (C) qRT-PCR and Western blot analysis of Rpp29 mRNA and ATRX protein levels, respectively, in HeLa HI 1-1 cells after shRNA knockdown. For qRT-PCR, results are the average of three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (D) The transgene diagram shows the location of the primer pairs used for qPCR analysis of the ChIP results in HeLa HI 1-1/H3.3-YFP cells. (E) ChIP analysis of H3.3-YFP incorporation into the transgene array after knockdowns in HeLa HI 1-1/H3.3-YFP cells. Results are the average of at least three independent experiments, and p values were calculated by comparing pLKO.1 (blue bar) to the shRNA data sets using unpaired t test: *p ≤ 0.05 and **p ≤ 0.01. (F) Representative Western blot of high-salt extraction assay showing H3.3-YFP and H3.3 levels detected using anti-GFP and anti-H3.3 antibodies. Graphs show measurements of (I) H3.3-YFP with anti-GFP antibody, (II) H3.3-YFP with anti-H3.3 antibody, and (III) H3.3 with anti-H3.3 antibody. Results are the average of at least three independent experiments, and p values were calculated using unpaired t test: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (G) ChIP analysis of H3.3-YFP incorporation into the transgene array in HeLa HI 1-1/H3.3-YFP cells after first detergent extracting and vortexing isolated nuclei.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814222&req=5

Figure 8: Rpp29 represses histone H3.3 chromatin assembly. (A) Western blot shows levels of endogenous H3.3 and H3.3-YFP in the HeLa HI 1-1/H3.3-YFP (lane 1) and HeLa HI 1-1 (lane 2) cell lines. (B) Diagram of the steps undertaken in the high-salt extraction and chromatin immunoprecipitation protocols. (C) qRT-PCR and Western blot analysis of Rpp29 mRNA and ATRX protein levels, respectively, in HeLa HI 1-1 cells after shRNA knockdown. For qRT-PCR, results are the average of three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (D) The transgene diagram shows the location of the primer pairs used for qPCR analysis of the ChIP results in HeLa HI 1-1/H3.3-YFP cells. (E) ChIP analysis of H3.3-YFP incorporation into the transgene array after knockdowns in HeLa HI 1-1/H3.3-YFP cells. Results are the average of at least three independent experiments, and p values were calculated by comparing pLKO.1 (blue bar) to the shRNA data sets using unpaired t test: *p ≤ 0.05 and **p ≤ 0.01. (F) Representative Western blot of high-salt extraction assay showing H3.3-YFP and H3.3 levels detected using anti-GFP and anti-H3.3 antibodies. Graphs show measurements of (I) H3.3-YFP with anti-GFP antibody, (II) H3.3-YFP with anti-H3.3 antibody, and (III) H3.3 with anti-H3.3 antibody. Results are the average of at least three independent experiments, and p values were calculated using unpaired t test: *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. (G) ChIP analysis of H3.3-YFP incorporation into the transgene array in HeLa HI 1-1/H3.3-YFP cells after first detergent extracting and vortexing isolated nuclei.
Mentions: To determine whether Rpp29 regulates H3.3 nucleosome deposition, we knocked Rpp29 down in the HeLa cell line stably expressing H3.3-YFP (Figure 8A) and evaluated H3.3 incorporation into the inactive transgene array using chromatin immunoprecipitation (ChIP; Figure 8B is a diagram of the procedure). We previously reported that, in the HeLa cell line, H3.3 is significantly enriched in the inactive transgene array (Figure 8D is a diagram of the primer pairs) compared with the RC variant, H3.2 (Newhart et al., 2012). Here we show that Rpp29 knockdown significantly increases H3.3 incorporation into the array (Figure 8E, brown bars). Taken together, these results suggest that Rpp29 represses H3.3 chromatin assembly by down-regulating a transcriptional recruitment signal.

Bottom Line: Rpp29 is a protein subunit of RNase P.Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly.Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Wistar Institute, Philadelphia, PA 19104.

No MeSH data available.


Related in: MedlinePlus